Medical therapy for infection with the human immunodeficiency virus type 1 (HIV-1) has improved over the last several years. However, many patients still fail to respond to the available pharmacologic agents, due to drug resistance and other factors. In addition, the antivirals currently available are often not well tolerated by patients.
The currently available drugs for treating HIV-1 infection either attack the reverse transcriptase or protease enzymes of the virus. Such compounds have the problem of rapidly developing drug resistance due to rapid transformation of the infecting viral genome. However, despite the problem of rapid drug resistance such drugs have been used to treat HIV seropositive individuals to help prevent the progression of the infection into the clinical symptoms of acquired immunodeficiency syndrome (AIDS). Such drugs are also used to obtain regression of AIDS.
Infection of CD4+ T lymphocytes and monocyte-derived macrophages (MDM) by the human immunodeficiency virus type 1 (HIV-1) triggers the production and release of inflammatory mediators. Butera, S. T. et al., J Immunol 150, 625–34 (1993); D'Addario, M. et al., J Virol 64, 6080–9 (1990); Nakajima, K. et al., J Immunol 142, 531–6 (1989); Poli, G. et al. and Fauci, Semin Immunol 5, 165–73 (1993); Meltzer, Annu Rev Immunol 8, 169–194 (1990). These cytokines have been shown to participate in the host defense against HIV-1, but also contribute to the pathogenesis of HIV-1 infection. Fauci, A. S., Science 262, 1011–8 (1993). A subset of the cytokine family, the chemokines, are a group of small polypeptide molecules that attract and activate leukocytes via interactions with seven-transmembrane-domain G-protein-coupled-receptors (GPCR) Loetscher, P. et al., Adv Immunol 74, 127–80 (2000). The chemokines are divided into subgroups according to the position of the first two cysteine residues in their sequence. C-C chemokines act primarily on mononuclear cells, including macrophages and lymphocytes. While initially characterized by their ability to attract neutrophils, some C-X-C chemokines have been shown to attract activated T cells, act as angiogenic regulators, and to stimulate monocyte adherence. Gerszten, R. E. et al., Nature 398, 718–23 (1999); Strieter, R. M. et al., J Biol Chem 270, 27348–57 (1995); Ward, S. G. et al., Immunity 9, 1–11 (1998).
Chemokines and chemokine receptors have recently come to the forefront of HIV biology. In the past few years, it has been shown that the C-C chemokines RANTES, MIP-1α and MIP-1β suppress HIV replication Cocchi, F. et al., Science 270, 1811–1815 (1995); Coffey, M. J. et al., Am J Physiol 272, L1025–9 (1997), although under some circumstances these same chemokines can actually enhance HIV-1 replication. Dolei, A. et al., Aids 12, 183–190 (1998); Gordon, C. J. et al., J Virol 73, 684–94 (1999); Kelly, M. D. et al., J Immuno 160, 3091–5 (1998); Kinter, A. et al., Proc Natl Acad Sci USA 95, 11880–11885 (1998); Marechal, V. et al., J Virol 73, 3608–15 (1999); Margolis, L. B. et al., J Clin Invest 101, 1876–80 (1998); Moriuchi, H. et al., J Exp Med 187, 1689–97 (1998); Moriuchi, M. et al., J Clin Invest 102, 1540–1550 (1998); Schmidtmayerova, H. et al., Proc Natl Acad Sci USA 93, 700–704 (1996); Trkola, A. et al., J Virol 73, 6370–9 (1999). It is now well known that HIV-1 enters cells by binding first to the CD4 receptor, which induces a conformational change in gp120, and then to a second receptor, which exposes the fusogenic epitopes of gp41 and permits fusion with the host cell membrane and subsequent entry into the cell. It has been shown that the major co-receptor for T cell tropic HIV-1 is the C-X-C chemokine receptor CXCR4, the ligand for which is SDF-1α. Bleul, C. C. et al., Nature 382, 829–33 (1996); Bleul, C. C. et al., J Exp Med 184, 1101–9 (1996); Feng, Y. et al., Science 272, 872–7 (1996). The principal co-receptor for monocytotropic HIV-1 isolates has been identified as CCR5, a C-C chemokine receptor that has as its ligands RANTES, MIP-1α and MIP-1β. Alkhatib, G. et al., Science 272, 1955–1958 (1996); Choe, H. et al., Cell 85, 1135–48 (1996); Deng, H. et al., Nature 381, 661–6 (1996); Doranz, B. J. et al., Cell 85, 1149–58 (1996); Dragic, T. et al., Nature 381, 667–73 (1996). It has been demonstrated that individuals having mutations in both alleles for CCR5 are highly resistant to HIV infection. Dean, M. et al., Science 273, 1856–62 (1996); Liu, R. et al., Cell 86, 367–77 (1996); Samson, M. et al., Nature 382, 722–5 (1996). Further, some evidence indicates that persons heterozygous for a mutation in CCR5 have a delayed clinical course following HIV infection Dean, M. et al., Science 273, 1856–62 (1996); Samson, M. et al., Nature 382, 722–5 (1996); Smith, M. W. et al., Science 277, 959–65 (1997). A polymorphism in the gene for SDF-1α has also been associated with slower disease progression in HIV-1-infected individuals (Winkler, C. et al., Science 279, 389–93 (1998)), and SDF-1α blocks entry by CXCR4-using (X4) isolates of HIV. Bleul, C. C. et al., Nature 382, 829–33 (1996); Bleul, C. C. et al., J Exp Med 184, 1101–9 (1996); Oberlin, E. et al., Nature 382, 833–835 (1996). However, like RANTES, SDF-1α can actually potentiate HIV-1 replication under certain circumstances. Marechal, V. et al., J Virol 73, 3608–15 (1999).
The role that other chemokines play in HIV pathogenesis has been little studied. IL-8, the prototypical member of the C-X-C chemokine family, has been extensively characterized as a chemotactic factor for neutrophils. Loetscher, P. et al., Adv Immunol 74, 127–80 (2000); Yoshimura, T. et al., Proc Natl Acad Sci USA 84, 9233–7 (1987). Elevated levels of IL-8 have been detected in the serum and lungs of HIV-infected individuals. Denis, M. et al., AIDS Res Hum Retroviruses 10, 1619–27 (1994); Lipschik, G. Y. et al., Chest 104, 763–9 (1993); Matsumoto, T. et al., Clin Exp Immunol 93, 149–51 (1993). The presence of elevated levels of IL-8 in individuals infected with HIV-1 has led several groups to suggest that IL-8 plays a role in the pathogenesis of HIV-1 disease and infection with opportunistic infections, but little evidence has been found to support these claims.
Chemokine receptors are seven transmembrane domain-containing G-protein coupled receptors (GPCR) that transmit signals induced by a family of small, secreted polypeptides collectively known as chemokines. Chemokines attract and activate leukocytes and are divided into subgroups according to the position of the first two cysteine residues. Ward, S. G. et al., Immunity 9, 1–11 (1998). C-C chemokines act primarily on mononuclear cells, including MDM, lymphocytes, and eosinophils. Ward, S. G. et al., Immunity 9, 1–11 (1998). While initially thought to act principally on neutrophils, some C-X-C chemokines have been shown to attract activated T cells, act as angiogenic regulators, and stimulate monocyte adherence. Ward, S. G. et al., Immunity 9, 1–11 (1998); Strieter, R. M. et al, J Biol Chem 270, 27348–27357 (1995); Gerszten, R. E. et al., Nature 398, 718–723 (1999).
The C-X-C chemokine growth-regulated oncogene-alpha (GRO-α), also called melanoma growth stimulatory activity (MGSA), was initially identified as an autocrine growth factor for malignant melanoma cells. Richmond, A. et al., J Cell Physiol 129, 375–384 (1986). Subsequent studies have shown that the receptor for GRO-α is CXCR2, and GRO-α attracts cells that express this receptor, including both neutrophils and dendritic cell. Anisowicz, A. L. et al., Proc Natl Acad Sci USA 84, 7188–7192 (1987); Moser, B. et al, J Exp Med 171, 1797–1802 (1990); Ahuja, S. K. et al., J Biol Chem 271, 20545–20550 (1996). GRO-α also has been demonstrated to have direct angiogenic activity in several in vivo assays, and to activate orf 74 of the Kaposi's sarcoma herpesvirus (KSHV), a GPCR that has been implicated in the transformation and angiogenic phenotype of KS lesions. Strieter, R. M. et al., J Biol Chem 270, 27348–27357 (1995); Luan, J. et al., J Leukoc Biol 62, 588–597(1997); Gershengorn, M. C. et al., J Clin Invest 102, 1469–1472 (1998); Bais, C. et al., Nature 391, 86–89 (1998). GRO-α and the two other GRO chemokines, GRO-β and GRO-γ, are encoded by distinct genes, are 88% identical at the amino acid level, signal through CXCR2, and are thought to be largely functionally redundant. Ahuja, S. K. et al., J Biol Chem 271, 20545–20550 (1996); Luan, J. et al., J Leukoc Biol 62, 588–597 (1997).
Aberrant function of both infected and uninfected MDM has been implicated in the pathogenesis of HIV dementia and AIDS-associated opportunistic infections. Merrill, J. E., Dev Neurosci 14, 1–10 (1992); Shiratsuchi, H. et al., J Clin Invest 93, 885–891 (1994); Koziel, H. Q. et al., J Clin Invest 102, 1332–1344 (1998); Gartner, S., Science 287, 602–604 (2000). MDM infected with HIV-1 are known to produce a host of inflammatory mediators, including TNF-α, IL-1, and IL-6, less than 48 hours after infection. Merrill, J. E. et al., J Virol 63, 4404–4408 (1989); Clouse, K. A. et al., J Immunol 147, 2892–2901 (1991); Herbein, G. et al., Clin Exp Immunol 95, 442–449 (1994); Borghi, P. et al., J Virol 69, 1284–1287 (1995); Poli, G. et al., Cambridge, Mass.: Blackwell Scientific, pp. 421–449 (1995). In addition, macrophages exposed to HIV-1 contribute to the death of T lymphocytes by TNF-α- and FasL-dependent pathways. Badley, A. D. et al., J Exp Med 185, 55–64 (1997); Herbein, G. et al., Nature 395:189–194 (1998). Following infection, macrophages also produce the C-C chemokines RANTES, MIP-1α, and MIP-1β. Canque, B. et al., Blood 87, 2011–2019 (1996); Schmidtmayerova, H. et al., Proc Natl Acad Sci USA 93, 700–704 (1996). These three chemokines inhibit HIV replication by nature of their ability to ligate the HIV co-receptor CCR5 and prevent HIV entry, both by blocking binding sites and inducing receptor internalization. Cocchi, F. et al., Science 270, 1811–1815 (1995); Trkola, A. et al., Nature 384, 184–187 (1996); Wu, L. et al., Nature 384, 179–183 (1996); Coffey, M. J. et al., Am J Physiol 272, L1025–1029 (1997); Mack, M. et al., J Exp Med 187, 1215–1224 (1998). Subsequent reports have demonstrated that RANTES can also stimulate the replication of X4 HIV by activating, and increasing virion attachment to, target cells. Kinter, A. et al., Proc Natl Acad Sci USA 95, 11880–11885 (1998); Dolei, A. et al., Aids 12, 183–190 (1998); Gordon, C. J. et al., J Virol 73, 684–694 (1999); Trkola, A. et al., J Virol 73, 6370–6379 (1999). Similarly, the CXCR4 ligand SDF-1 can both prevent X4 HIV entry by inducing receptor internalization and stimulate HIV proviral gene expression. Bleul, C. C. et al., Nature 382, 829–833 (1996); Oberlin, E. et al., Nature 382, 833–835 (1996); Amara, A. et al., J Exp Med 186, 139–146 (1997); Marechal, V. et al., J Virol 73, 3608–3615 (1999). While much is known about the role of RANTES, MIP-1α, and MIP-1β in HIV pathogenesis, relatively little is known about the role of other chemokines produced by HIV-infected leukocytes.
Kaposi's sarcoma (KS) is one of the two most common neoplasms associated with HIV-1 infection and is difficult to treat clinically. Boshoff, C. et al., Adv Cancer Res 75, 57–86 (1998). KS is characterized by complex spindle cell neoplasms, composed of endothelial cells, fibroblasts, dermal dendrocytes, and inflammatory cells. Friedman-Kien, A. E., J Am Acad Dermatol 5, 468–71 (1981); Gottlieb, G. J. et al., Hum Pathol 13, 882–92 (1982); McNutt, N. S. et al., Am J Pathol 111, 62–77 (1983). In the early stages of disease, KS is not a monoclonal tumor, but an angioproliferative lesion driven by inflammatory cytokines. Fiorelli, V. et al., Blood 91, 956–67 (1998); Regezi, J. A. et al., Am J Pathol 143, 240–9 (1993). Although the origin of KS is still controversial, mounting evidence suggests that endothelial cells are the progenitors of the KS spindle cells. Boshoff, C. et al., Nat Med 1, 1274–8 (1995); Boshoff, C. et al., Adv Cancer Res 75, 57–86 (1998). Since Chang and coworkers first reported that over 90% of AIDS-KS tissue samples were positive for a new member of the γ-herpesvirus family, the Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus-8 (HHV-8), KSHV is believed to be necessary for the development of KS. Chang, Y. et al., Science 266, 1865–9 (1994); Gallo, R. C., Science 282, 1837–9 (1998); Mesri, E. A., Blood 93, 4031–3 (1999). KSHV infects endothelial cells and is found in spindle cells, and prior KSHV infection clearly predisposes for the onset of KS in AIDS patients. Boshoff, C. et al., Nat Med 1, 1274–8 (1995); Gao, S. J. et al., N Engl J Med 335, 233–41 (1996); Rezza, G. et al., J Natl Cancer Inst 91, 1468–74 (1999).
KSHV contains multiple open reading frames (ORF) encoding cellular homologs, including ORF 74, a GPCR most similar to CXCR2. Arvanitakis, L. et al., Nature 385, 347–50 (1997). Constitutive signaling by ORF 74 in NIH3T3 cells results in transformation of these cells, and transgenic mice expressing ORF 74 develop a disease with striking similarity to KS. Bais, C. et al., Nature 391, 86–89 (1998); Yang, T. Y. et al., J Exp Med 191, 445–54 (2000). The ligands for CXCR2, including IL-8, bind to ORF 74 with high affinity and activate signaling through this receptor. Gershengorn, H. C. et al., J Clin Invest 102, 1469–72 (1998). However, the biological ramifications of such signaling have not been clear.
While HIV-1 infection alone is not sufficient for the development of KS, AIDS-associated KS is more aggressive, disseminated, and resistant to treatment than the other forms of KS, including post-transplant KS. Buchbinder, A. et al., Curr Opin Oncol 4, 867–74 (1992); Friedman-Kien, A. E. et al., Ann Intern Med 96, 693–700 (1982); Strathdee, S. A. et al., Aids 10, S51–7 (1996). Therefore, immunosuppression alone cannot explain the frequent presentation of KS prior to immunosuppression, the greater prevalence of KS in AIDS patients than in other immunosuppressed individuals, and the association of KS with HIV-1, but not HIV-2, infection in West Africa. Ariyoshi, K. et al., J Hum Virol 1, 193–9 (1998); Beral, V. et al., Lancet 335, 123–8 (1990); Mocroft, A. et al., Arch Intern Med 158, 491–7 (1998); Poznansky, M. C. et al., Bmj 311, 156–8 (1995). Thus, there has been much interest in HIV-related factors which might potentiate the development of KS. The HIV-1 transactivator protein Tat has been clearly implicated in the growth of KS cells, and in combination with basic fibroblast growth factor (bFGF), is believed to drive the formation of lesions. Ensoli, B. et al., Nature 345, 84–6 (1990); Ensoli, B. et al., Nature 371, 674–80 (1994). Extracellular Tat acts in a paracrine fashion on KS cells by binding to integrin receptors. Barillari, G. et al., Proc Natl Acad Sci USA 90, 7941–5 (1993); Ensoli, B. et al., J Virol 67, 277–87 (1993). Thus, Tat is the HIV-1 protein with the strongest link to the pathogenesis of KS.
It would thus be desirable to provide a method of controlling viral replication in a host cell comprising inhibiting the biological action of chemokines in stimulating viral replication. Such methods could involve eliminating or sequestering the chemokines or alternately, blocking chemokine receptors so that the chemokines cannot bind.
It would be further desirable to provide methods for treating a patient with a viral infection. Preferably such methods will inhibit or suppress viral replication resulting in the prevention, delay or abatement of the symptoms associated with a viral infection.